In the last two years we have shown that: 1) Wwox, Ap23, and ErbB4 are independent markers of tamoxifen resistance. Reduced Wwox expression was better than PR in prediction of resistance, especially in high-risk patients, and nuclear Ap23 expression was better than Her2, especially in low-risk patients;2) ErbB4 loss was an independent marker of tamoxifen resistance when adjusted for other significant predictors;3) in vitro studies of tamoxifen resistant cells confirmed that Ap23 was bound by Wwox in the cytoplasm, released into the nucleus in Wwox negative, tamoxifen resistant cells and was a nuclear activator of HER2;4) >800 invasive breast cancers on Tissue Micro Arrays (TMAs) were classified into specific subtypes: triple negative tumors (TN: ER, PR, HER2 negative, basal-like tumors) showed frequent expression of EGFR, CK5/6 and AP23 and frequent loss of Fhit and Wwox, suggesting that reduced Fhit and Wwox expression have roles in pathogenesis of basal differentiation in breast cancer. Alteration of expression of Fhit and Wwox occured in ~90% of the basal-like/TN breast cancers and may contribute to defects in DNA repair, as observed in BRCA1- deficient cancers. DNA damage response (DDR) proteins (3H2AX, pChk2, p53) were expressed highly significantly more in TN and basal-like tumors. Thus, DDR checkpoint proteins could be targets for treatment of these cancers. Specific subtype breast cancer cell lines, for mechanistic studies, and TMAs with cores from primary and metastatic cancers with linked treatment and outcome data, for in vivo studies, will be used in experiments outlined in the following Aims: 1. Wwox effectors in breast cancer in vitro: a) using breast cancer cells of luminal A, HER2+ and TN/Basal subtypes, identify Wwox interactor proteins in specific subtypes, after infection with AdenoWWOX, isolation of the Wwox complex, followed by mass spectrometry identification of proteins in the complex;b) examine expression of candidate interactors in specific breast cancer subtypes in cell lines and tissues;2. DNA damage response checkpoint inhibitors in breast cancer in vitro: a) Chk1 and Parp1 inhibitors will be tested for effect on specific breast cancer subtypes in tissue culture;b) Chk1 and Parp1 inhibitors, in combination with cytotoxic chemotherapeutic drugs, will be tested for effect on cytotoxicity of specific breast cancer subtypes;3. Association of activated DDR checkpoint in breast cancers with treatment outcome: divide tumors on TMAs into subtypes based on ER, PR, HER2, EGFR, CK5,6 status;assess status of the DDR checkpoint proteins in tumors of each subtype and correlate markers with treatment and outcome;4. Conditional mouse Wwox knockdown X Fhit knockout. The mouse cross will be examined for susceptibility to mammary gland cancer and mechanisms involved i development of normal and neoplastic glands. 1 PUBLIC HEALTH RELEVANCE: We find that triple-negative/basal-like breast cancers, the typically aggressive breast cancers that commonly afflict young women and women of African origin, preferentially show evidence of expression of activated checkpoint proteins;also triple-negative/basal-like cancers express reduced Fhit and Wwox, proteins involved in protection of genome integrity, with Fhit loss resulting in strong checkpoint activation and resistance to cisplatin and paclitaxel. BRCA1 and 2 mutant breast cancers are usually of the basal-like subtype and when tested in the laboratory, are highly susceptible to killing by inhibitors of the DNA damage response checkpoint. We will determine if basal-like breast cancer cells and other Fhit/Wwox-deficient breast cancer subtypes are, like BRCA1/2-deficient cells, susceptible to killing by checkpoint inhibitors. Chemotherapy is currently the only option for these cancers and might become a better option if targeted inhibition of DNA repair pathways were combined with DNA damaging agents, such as carboplatin.